An analysis of phosphorylation sites in protein kinases from Leishmania.

Protein kinases are promising drug targets for Leishmaniasis. We have evaluated the phosphorylation potential of protein kinases in different species and strains of Leishmania. Phosphorylation potential of serine, threonine and tyrosine residues of kinases in Leishmania parasite were studied. The results indicate that some species specific residues of serine, threonine and tyrosine have a phosphorylation potential of 1 suggesting that these residues are important target sites in protein kinases based anti-leishmanial therapies.

Analysis of proteins involved in the production of MAA׳s in two Cyanobacteria Synechocystis PCC 6803 and Anabaena cylindrica.

Mycosporine- like amino acids (MAAs) are small (<400Da), colourless, water soluble compounds composed of cyclohexenone or cyclohexinimine chromophere conjugated with the nitrogen substituent of amino acid or its amino alcohol. These compounds are known for their UV- absorbing role in various organisms and seem to have evolutionary significance. The biosynthesis of MAAs is presumed to occur via the first part of shikimate pathway. In the present work two cyanobacteria Synechocystis PCC 6803 and Anabaena cylindrica were tested for their ability to synthesize MAAs and protein involved in the production of MAAs. It was found that protein sequence 3-phosphoshikimate 1-carboxyvinyltransferase is involved in producing mycosporine glycine in Synechocystis PCC 6803 and 3-dehydroquinate synthase is involved for producing shinorine in Anabaena cylindrica. Phylogenetic and bioinformatic analysis of Mycosporine like amino acid producing protein sequence of both cyanobacterial species Synechocystis PCC 6803 and Anabaena cylindrica provide a useful framework to understand the relationship of the different forms and how they have evolved from a common ancestor. These products seem to be conserved but the residues are prone to variation which might be due the fact that different cyanobacteria show different physiological process in response of Ultraviolet stress.

A comprehensive analysis of LACK (Leishmania homologue of receptors for activated C kinase) in the context of Visceral Leishmaniasis.

The Leishmania homologue of activated C kinase (LACK) a known T cell epitope from soluble Leishmania antigens (SLA) that confers protection against Leishmania challenge. This antigen has been found to be highly conserved among Leishmania strains. LACK has been shown to be protective against L. donovani challenge. A comprehensive analysis of several LACK sequences was completed. The analysis shows a high level of conservation, lower variability and higher antigenicity in specific portions of the LACK protein. This information provides insights for the potential consideration of LACK as a putative candidate in the context of visceral Leishmaniasis vaccine target.

Computational protein structure modeling and analysis of UV-B stress protein in Synechocystis PCC 6803.

This study focuses on Ultra Violet stress (UVS) gene product which is a UV stress induced protein from cyanobacteria, Synechocystis PCC 6803. Three dimensional structural modeling of target UVS protein was carried out by homology modeling method. 3F2I pdb from Nostoc sp. PCC 7120 was selected as a suitable template protein structure. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in modeled UV-B stress protein. The top five probable ligand binding sites were predicted and the common binding residues between target and template protein was analyzed. It has been validated for the first time that modeled UVS protein structure from Synechocystis PCC 6803 was structurally and functionally similar to well characterized UVS protein of another cyanobacterial species, Nostoc sp PCC 7120 because of having same structural motif and fold with similar protein topology and function. Investigations revealed that UVS protein from Synechocystis sp. might play significant role during ultraviolet resistance. Thus, it could be a potential biological source for remediation for UV induced stress.

Expression and in silico characterization of Phenylalanine ammonium lyase against karnal bunt (Tilletia indica) in wheat (Triticum aestivum).

To investigate the lignifications process and its physiological significance under Karnal Bunt (KB), the changes in enzymes responsible for lignifications likes, phenylalanine ammonia lyase (PAL), were determined in resistant (HD-29) and susceptible genotype (WH-542) of wheat during different developmental stages. The PAL gene was cloned and sequenced. The expression of PAL gene was measured by means of semi-quantitative RT-PCR. The enzyme was expressed constitutively in both the susceptible and resistant genotype. However, the activity was higher in all the developmental stages of resistant genotype, indicating that this genotype has a significant higher basal level of these enzymes as compared to the susceptible line and could be used as marker(s) to define KB resistance. The activity of PAL was significantly higher in WSv stage (Z=16). Structural comparisons based on alignments of all the protein sequences using the clustal W program and searches for conserved motifs using the MEME program have revealed broad conservation of main motifs characteristic of the plant PAL. MSA and phylogenetic analyses of different plants PAL demonstrate that all PAL cluster divided in to two main cluster. The PAL also possesses a specific consensus sequences [GS]- [STG]-[LIVM]-[STG]-[SAC]-S-G-[DH]-L-x-[PN]-L-[SA]-x(2,3)-[SAGVTL]. The pathway might be associated with the enhancement of structural defense barrier due to lignifications of cell wall as evident from the enhanced synthesis of lignin in all the stages of resistant genotype. Our results clearly indicate the possible role of enzymes of Phenyl propanoid pathway metabolism provides genotype and stage dependant structural barrier resistance in wheat against KB.

Leishmania donovani: CD2 biased immune response skews the SAG mediated therapy for a predominant Th1 response in experimental infection.

We have evaluated the effect of combining CD2 with conventional antimonial (sb) therapy in protection in BALB/c mice infected with either drug sensitive or resistant strain of Leishmania donovani with 3×10(7) parasites via-intra-cardiac route. Mice were treated with anti CD2 adjunct SAG sub-cutaneously twice a week for 4 weeks. Assessment for measurement of weight, spleen size, anti-Leishmania antibody titer, T cell and anti-leishmanial macrophage function was carried out day 0, 10, 22 and 34 post treatments. The combination therapy was shown boosting significant proportion of T cells to express CD25 compared to SAG monotherapy. Although, the level of IFN-γ was not statistically different between combination vs monotherapy (p=0.298) but CD2 treatment even alone significantly influenced IFN-γ production than either SAG treatment (p=0.045) or with CD2 adjunct SAG treatment (p=0.005) in Ld-S strain as well as in Ld-R strain. The influence of CD2 adjunct treatment was also documented in anti-leishmanial functions in macrophages. As shown, the super-oxide generation began enhancing very early on day 10 after SAG treatment with CD2 during which SAG action was at minimum. Interestingly, the super-oxide generation ability remained intact in macrophage after treatment with immuno-chemotherapy even in mice infected with Leishmania resistant strain. Unlike SAG treatment, treatment of SAG with CD2 also led to production of nitric oxide and TNF-α, resulting in resulting in most effective clearance of L. donovani from infected macrophages. Our results indicate that CD2, which can boost up a protective Th1 response, might also be beneficial to enable SAG to induce Macrophages to produce Leishmanicidal molecules and hence control the infection in clinical situation like Kala-azar. Drug resistance is the major impedance for disease control but the encouraging results obtained after infecting mice with resistant strain of the parasite strongly imply that this drug can be effective even in treating resistant cases of Kala-azar.

Antimony resistance during Visceral Leishmaniasis: A possible consequence of serial mutations in ABC transporters of Leishmania species.

Visceral Leishmaniasis is a macrophage associated disorder for the treatment of which antimony based drugs like SAG and SSG were the first choice in the recent past. The clinical value of antimony therapy is now declined against VL because increasing cases of Sodium Antimony Gluconate (SAG) resistance have reached outstanding proportion in Bihar, India. Within this context we looked into the protein sequences of ABC transporters of Leishmania spp associated with Visceral Leishmaniasis that are known to play a crucial role in the development of multidrug resistance (MDR). Our studies consisting of ClustalW, Phylogeny and TCOFFEE have pinpointed that ABC transporters have enormously diverged during the process of evolution even within the identical species strains resulting in insignificant homology and subdued conservation amongst the aminoacid residues. Moreover these amino acid residues remain susceptible to mutations in evolutionary era as indicated by high frequency of variations by the variability studies. Hence we predict that during the process of evolution a series of frequent mutations might have led to changes in the ABC transporters favorable to effluxing the drug thereby making the Leishmania species prone to resistance against the efficient first line drug SAG, used for combating VL. This selection has made them to survive efficiently in the adverse circumstances of antimony based antileishmanial therapy regime.

A gp63 based vaccine candidate against Visceral Leishmaniasis.

Visceral leishmaniasis is a macrophage associated disorder which leads to a profound decrease in the natural immunotherapeutic potential of the infected subjects to combat the disease. The major surface glycoprotein gp63 has been found to be a significant vaccine candidate against visceral leishmaniasis. The current study addresses the levels of similarity and identity in the gp63 obtained from different species of Leishmania viz donovoni, chagasi and infantum linked to the cause of visceral leishmaniasis. The results from BLAST, Phylogram and Cladogram studies indicate significant identity, similarity and conservation of important residues in the protein which lead us to conclude that a common gp63 based vaccine can be used as a therapeutical tool against visceral leishmaniasis caused by different species strains of leishmania.

Leishmania donovani: role of CD2 on CD4+ T-cell function in Visceral Leishmaniasis.

In this study, we investigated whether alteration in the CD2 mediated coordination of an immune response was associated with down regulation of CD4 associated Th1 cell response during Visceral Leishmaniasis (VL). Leishmania donovani (Ld) infection in VL patients markedly reduced expression of CD2 cell surface antigen on CD4+ cells. T-cells of VL patients were mostly in G0/G1 stage of the cell cycle (98.20%) with little or no activity of protein kinase C-alpha (PKC-alpha) isoform. However, pre-incubation with activating anti-CD2 monoclonal antibody (MAb) resulted in a corresponding increase up to 2.52-fold in T-cells of G2/M population supported by both activity and expression of PKC-alpha isoform. Furthermore, we observed that co-incubation of T-cell with anti-CD2 increased the lymphocyte-blast population in patients in whom the CD4 cells became more antigen responsive (CD4+ CD69+ cells). Consistent with these observations, it was shown that 59.3% of CD4 cells from patients responded to Ld by producing IFN-gamma. Even in the culture condition, when the T-cells from patients were depleted of APC, IFN-gamma production was noticed after CD2 activation. On the other hand, IL-4 production became low in the anti-CD2 antibody supplemented peripheral blood mononuclear cells (PBMNCs) culture. These findings imply that infection with L. donovani induces less CD2 on the surface of CD4+ T-cells, which once activated orchestrate the protective IFN-gamma dominant host defense mechanism via PKC-mediated signal transduction and cell cycle.

The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function.

Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.